The present invention relates to a novel device for small sample preparation using tubes and columns, such as capillaries or pipette tips, in which particles of a separation medium, such as particles of a chromatography material used for sample preparation, are directly embedded in the solid material composing the tubes or columns. Most small sample preparation methods currently available cannot prevent sample loss and they are not well suited for small sample volumes. The present invention minimizes sample loss and is well suited for samples with volumes in nanoliters.
In the invention described herein, the desired sample, such as a sample composed of biomolecules, is brought in contact with particles of the separation medium embedded on the surface of the device comprised of tubes and columns, according to the present invention. The sample is separated, analyzed, purified or prepared for further analysis as the sample components come in contact with the separation medium embedded in the tubes or columns, according to the present invention. The tubes or columns described in the present invention can be capillaries, pipette tips, combinations thereof, or any other devices suited for the preparation and analysis of small samples with volumes from nanoliters to hundreds of milliliters. The present invention also describes a method for making tubes or columns that have particles of separation media embedded on their surface for sample preparation applications.
A number of methods for separating, purifying or preparing small biological samples currently exist. Yet, many of the available methods present significant disadvantages, which can be overcome by the current invention. Currently, sample preparation is performed using spin columns, filter and separation medium filled chromatography columns and even pipette tips filled or coated with separation media such as chromatography materials. Sample preparation using these available devices is performed through centrifugation, gravitation, vacuum suction, and pressure application or by syringe-based or pressure-based sample delivery through the columns or tips. Such columns are used for the separation and purification of small sample volumes from nanoliters to milliliters. The samples purified using these methods can be any type of samples such as samples containing biomolecules such as proteins, DNA, nucleic acids and other biological molecules. Many different types of separation media are used in currently available columns including but not limited to chromatography materials such as gel-filtration, affinity, ion-exchange, reverse-phase, and silica or modified-silica materials.
Although many different analytical methods for small sample separation and purification have been developed, a number of problems, such as the slow speed of the separation process and the loss of sample volume limit the quality of results obtained using these methods. For example, in spin columns and small sample chromatography columns, filters are used to hold the separation medium within the column such that separate filters are placed above and/or below the separation medium in the column. In such columns the sample flows through the filters, in addition to the separation media, before being collected or retrieved for further analysis. One of the main problems with filters is that they slow the rate at which the sample passes through the column and they result in the loss of sample on the filter material.
At present, most macro spin columns and micro spin columns contain a filter at their bottom end. The sample loss is especially significant when very small sample volumes are separated using currently available methods. In fact, currently available methods are not well suited for the separation of very small sample volumes in the nanoliters range. Since the concentration of biomolecules in micro volume samples is so small, the retention of molecules on the filter can result in significant loss of the total sample volume. Also, since the volume of the filter is often as large as the volume of the micro volume sample itself, the separation or chromatography process is adversely affected due to the large volume of filter material through which the sample must pass during the separation process.
The filter material may also absorb proteins or biomolecules from the sample, resulting in lower than desirable sample recovery. Also, the filter material may behave differently in different elution media, subsequently interfering with both the quality of the separation process and the volume of the sample retained.
There are some filter-free columns that are currently available for sample preparation. In the most commonly available versions, such columns rely on a solid support matrix in which the separation medium is embedded. The combination of the solid matrix and the separation material is then adhered to a column or pipette tip to create a plug or coating through which the sample passes for sample preparation. In currently available technologies where a plug of the solid matrix and separation material is used, sample flow through the plug is slow, limiting the rate at which samples can be prepared. Also, sample loss in the plug limits the use of such technology for the preparation of very small samples.
In the present invention we describe a sample preparation device consisting of one or more tubes or columns where at least one of said tubes or columns contains particles of a separation medium directly embedded in the material comprising said tube or column. Said tubes or columns may be closed or open at one or both ends to impede or permit the flow of the sample through the tube or column. Said tubes or columns can be composed of any polymer materials. Thus, the sample preparation device described in the present invention is free of any filters or solid matrices that can potentially slow the rate of sample preparation and result in sample loss. Furthermore, the sample preparation device described in the present invention can be created such that it is suited for the separation and preparation of very small sample volumes, in the nanoliter range. The separation medium used in the present invention can consist of one or more types of different separation media such as chromatography materials such as gel-filtration, ion-exchange, reverse-phase, and silica or modified silica media.
Once desired components of a sample, such as biomolecules, bind to the separation media embedded in the tubes or columns, the sample components can then be eluted from the separation media using different solvents. The device described in the present invention is also very well suited for automated processes such as high throughput screening.
The various features of novelty, which characterize the invention, are pointed out with particularity in the claims annexed to and forming a part of this disclosure. For a better understanding of the invention, its advantages and objects, reference is made to the accompanying drawings and descriptive matter in which a preferred embodiment of the invention is illustrated.